Voltage-activated Na(+) channels contain one distinct α-subunit. In the brain NaV 1.1, NaV 1.2, NaV 1.3 and NaV 1.6 are the four most abundantly expressed α-subunits. The antiepileptic drugs (AEDs) carbamazepine, phenytoin and lamotrigine have voltage-gated Na(+) channels as their primary therapeutic targets. This study provides a systematic comparison of the biophysical properties of these four α-subunits and characterizes their interaction with carbamazepine, phenytoin and lamotrigine. Na(+) currents were recorded in voltage-clamp mode in HEK293 cells stably expressing one of the four α-subunits. NaV 1.2 and NaV 1.3 subunits have a relatively slow recovery from inactivation, compared with the other subunits and NaV 1.1 subunits generate the largest window current. Lamotrigine evokes a larger maximal shift of the steady-state inactivation relationship than carbamazepine or phenytoin. Carbamazepine shows the highest binding rate to the α-subunits. Lamotrigine binding to NaV 1.1 subunits is faster than to the other α-subunits. Lamotrigine unbinding from the α-subunits is slower than that of carbamazepine and phenytoin. The four Na(+) channel α-subunits show subtle differences in their biophysical properties, which, in combination with their (sub)cellular expression patterns in the brain, could contribute to differences in neuronal excitability. We also observed differences in the parameters that characterize AED binding to the Na(+) channel subunits. Particularly, lamotrigine binding to the four α-subunits suggests a subunit-specific response. Such differences will have consequences for the clinical efficacy of AEDs. Knowledge of the biophysical and binding parameters could be employed to optimize therapeutic strategies and drug development.